Antifungal genome mining and genetics in filamentous actinomycete bacteria isolated from local soils
Date Submitted
2017
Research Mentor and Department
Dr. Joshua A.V Blodgett, Biology
Restricted/Unrestricted
Unrestricted
Abstract
Actinomycetes are gram positive, filamentous bacteria that produce useful antibiotics, antitumor agents, and agricultural products. A series of enrichments were undertaken to isolate actinomycetes from local soils, varying enrichment media, antibacterials, and soil treatments (including heat and CaCO3). Isolates were characterized by 16S rDNA sequencing, phenotypic and morphological observations, and antibiotic production. The genetic tractability of select isolates was analyzed using a panel of integrating vectors derived from ɸC31, ɸBT1, and OzzyJ phage using intergeneric conjugation. Further, a semi-degenerate multiplex PCR assay to detect ɸBT1 genomic integrants was designed and tested for the first time. Finally, PCR screens were used to test if the isolates genetically encode for the production of Polycyclic Tetramate Macrolactams (PTM), a common class of antifungal natural products. We designed and tested PCR screens in silico that probed specific PTM biosynthetic genes in order to predict PTM chemical variability arising from gene cluster diversity. PTM production from positive isolates was assayed using coupled liquid chromatography-mass spectrometry (LC-MS). Our results indicate that we have isolated a variety of Actinomycetes, many of whom produce antifungal and antibacterial compounds, which are genetically tractable with a subset predicted to produce PTM compounds.